1. Local Actin-Dependent Endocytosis Is Zygotically Controlled to Initiate Drosophila Cellularization 
Anna Marie Sokac, Eric Wieschaus 
Developmental Cell 
Volume 14, Issue 5, Pages (May 2008)
DOI: /j.devcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Cell Cycle Progression Regulates Endocytosis at Early Mitotic Cycles
(A) Furrow dynamics at mitotic cycles (DNA, blue; somatic buds, jagged, black lines). The cortex is maximally displaced when metaphase furrows regress (purple arrow).
(B) Cross-sections taken at an early mitotic cycle show that few Amph (green) tubules (arrows) extend from somatic bud margins (Septin, red) at interphase, that many tubules extend from metaphase furrow tips at prophase/metaphase, and that no tubules extend from regressing metaphase furrows at telophase. DNA (blue) shows the phase of the cell cycle.
(C–E) Time-lapse cross-sections after perivitelline injection of Alexa 488-WGA. The 00:00 (min:s) time point was set relative to the start of image acquisition. (C) When injected at interphase, Alexa 488-WGA concentrates at somatic bud margins (arrowheads). As the embryo enters prophase, vesicles are released from the margins (arrows). (D) When injected at the interphase/prophase transition, Alexa 488-WGA labels ingressing furrows (arrowheads). Vesicles (arrows) are released from furrow tips. (E) When injected at interphase, Alexa 488-WGA patches blur as mitosis progresses. The first frame marks the onset of cortical displacement at late anaphase/telophase, when metaphase furrows regress and dramatic endocytosis ensues (arrows).
See Movies S2–S4. The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2 Local Endocytosis Occurs at Forming Cellularization Furrows
(A) Furrow dynamics at cellularization (DNA, blue; somatic buds, jagged, black lines; furrow canals, red).
(B–D) Cross-sections after perivitelline injection of Alexa 488-WGA. (B) Time-lapse images show that Alexa 488-WGA concentrates at somatic bud margins (arrowheads). Vesicles bud as cellularization furrows form. The 00:00 (min:s) time point was set relative to vesicle budding. See Movie S7. (C) Perivitelline injection of Alexa 546-WGA (red), followed by a 15 min chase and fixation. The embryo expresses FYVE-GFP (green). WGA binds the embryo surface immediate to injection site. The WGA label lengthens basally between nuclei (DNA, blue) as furrows ingress. WGA vesicles incorporate into early endosomes (arrowhead). (D) Higher-magnification view of the early endosome indicated in (B) by the arrowhead shows that Alexa 546-WGA (red) and FYVE-GFP (green) colocalize.
The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3 Local Endocytosis Is Regulated at Cellularization
(A) Cross-sections show that many Amph (green) tubules (arrows) extend from incipient furrow tips (Septin, red). At a furrow length of 5 μm, furrow canals are assembled (level of the black arrowhead), and few tubules are seen.
(B) The projected Z-section shows an Amph (green) tubule (arrows) extending from the furrow tip (Septin, red; level of the black arrowhead).
(C) The number of Amph tubules in wild-type embryos during cellularization. Each point represents one embryo with 160–180 furrows analyzed. Tubules are not detected at a furrow length ≥ 5 μm.
(D) Sequential planes in a Z-stack (moving deeper from left to right, 0.5 μm step) show an Amph structure in cross-section (arrow). The Amph structure appears as a circle, consistent with it being a tubule.
The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4 Nullo Regulates Endocytosis at Cellularization
(A) Cross-sections of nulloX embryos show that many Amph (green) tubules (arrows) extend from incipient furrow tips (Septin, red). At a furrow length of 5 μm (level of the black arrowhead), Amph tubules persist.
(B) Time-lapse cross-sections of a nulloX embryo after perivitelline injection of Alexa 488-WGA. Alexa 488-WGA concentrates at somatic bud margins (arrowheads). Budding vesicles distend into membrane tubules (arrows) and persist for up to several minutes before release. The 00:00 (min:s) time point was set relative to vesicle budding. See Movie S8.
(C) The number of Amph tubules in wild-type (black) versus nulloX (red) embryos during cellularization. Each point represents one embryo with 160–180 furrows analyzed. Tubules persist at a furrow length ≥ 5 μm in nulloX.
(D) The number of Amph tubules at permissive (black) versus restrictive (red) temperatures in shits1 embryos during cellularization, quantified as in (C). Tubules persist at a furrow length ≥ 5 μm at the restrictive temperature.
(E and F) Cross-sections show Amph tubules in shits1 embryos. (E) At the permissive temperature, few tubules are seen at a furrow length ≥ 5 μm. (F) At the restrictive temperature, tubules persist at a furrow length ≥ 5 μm.
The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5 Nullo Regulates Cortical F-Actin
(A–D) (A and C) Cross-sections of F-actin in comparably staged cellularizing embryos. Images were collected at the same settings for wild-type versus nullo manipulation. (A) nulloX furrow canals show reduced F-actin levles. (B) F-actin in wild-type (black) versus nulloX (red) furrow canals, measured by the intensity of phalloidin staining. Each point represents one embryo with 75–100 furrow canals analyzed. Error bars show standard deviation. (C) Ectopic Nullo furrow canals show increased actin levles. (D) F-actin in wild-type (black) versus ectopic Nullo (red) furrow canals, quantified as in (B).
(E and F) F-actin (red) in an imaginal wing disc expressing a stripe of ectopic Nullo (green). (E) The projected Z-section shows that F-actin is increased at the basal-lateral cortex of Nullo-expressing cells (A, apical; B, basal). (F) An en face image from a single basal plane of the disc showing that a stripe of F-actin increases in Nullo-expressing cells.
The scale bars are 5 μm in (A) and (C) and 20 μm in (F).
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6
F-Actin Disruption Impairs Endocytic Scission at Cellularization
(A) Cross-sections of Cyto-D versus DMSO control embryos. Amph tubules increase in number after Cyto-D treatment. At early cellularization multiple tubules may extend from each furrow tip (arrow).
(B) Time-lapse cross-sections after co-perivitelline injection of Alexa 488-WGA and Cyto-D. Some budding vesicles distend into tubules (arrow) that persist several minutes before release. The 00:00 (min:s) time point was set relative to vesicle budding.
See Movie S9. The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7
Persistent Amph Tubules Deplete Furrow Canal Components at Cellularization
(A and B) Cross-sections show DPATJ (green) and Amph (red) tubules. Arrowheads indicate the region of early endosomes. (A) DPATJ accumulates in wild-type furrow canals, but it is rarely seen in Amph tubules (arrows). Some DPATJ is seen in early endosomes. (B) DPATJ (green) accumulates in nulloX furrow canals, but it also enters Amph tubules (arrows). Increased levels of DPATJ are seen in nulloX early endosomes.
(C and D) En face images of furrow canals and cross-sections show DPATJ (green) and Myosin-2 (red). (C) DPATJ accumulates with Myosin-2 in wild-type furrow canals. (D) DPATJ and Myosin-2 are depleted from some nulloX furrow canals (arrowhead).
The scale bars are 5 μm.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions